Effect of carbon nanotubes on transepithelial resistance in barrier epithelial cells

poster abstractThe burgeoning of the nanotechnology industry has revolutionized engineering, medicine and the fashion industry amongst many other technologies. The arrays of products that are synthesized from nanomaterials include high definition TV and computer screens, artificial organs, as well as antibacterial food containers. With these novel applications there is a paralleled concern of the negative implications of nano-material contamination in our environment and food chains. We are interested in carbon nanotubes because they are the most abundantly occurring nanoparticles that are found in the workplace. We have recently conducted studies in barrier epithelial cells to show that long single-wall and multi-wall carbon nanotubes (CNTs) caused a decrease in the transepithelial electrical resistance, a measure of the barrier function ,of renal principal cells at very low concentrations (0.4 ng/cm2- 4 g/cm2). These results suggested that nanoparticles may also cause an effect on other barrier epithelial cells such as those lining the human digestive and respiratory tracks. After 48 hours of CNT exposure, to airway and colon cell lines, Calu-3 and t84 respectively, the calculated resistances were approximately half of the control monolayers’, indicating that the barrier function of the tissue had been compromised, while the cellular monolayer remained intact. We sought to determine the mechanism of action of the nanoparticles, by investigating the interaction of CNTs with model lipid membranes using a bilayer clamp amplifier. Measurements showed that the presence of nanoparticles caused transient disruptions in lipid membranes made of phosphatidylcholine lipids. Nanotubes also caused transient interruptions in the current allowed by the ion channel reporter (gramicidin A). These results began to elucidate the mode of action of the particles and indicated that it is important to develop a complete understanding of how nanoparticles interact with cells if we are to safeguard against changes that these materials will cause in vivo

Conductance of Ideally Cation Selective Channel Depends on Anion Type

poster abstractGramicidin A (gA) is a transmembrane, cation selective ion channel that has been used in many biophysical studies of lipid bilayers, in particular for investigations of lipid-protein interactions and membrane electrostatics. In addition, it was found that ionic interactions with neutral lipid membranes also affect the kinetics of gA channels. Here we report measurements of gA ion-channels for a series of sodium and potassium salts that show an anion-dependence of gA conductance. We find that gA conductance varies significantly with the anion type with ClO4 and SCN producing distinctly larger conductance values than Cl, F, and H2PO4. These results can provide new insights into ion-lipid membrane interactions and ion channel functions in general

Cation-selective channel is regulated by anions according to their Hofmeister ranking

Specificity of small ions, the Hofmeister ranking, is long-known and has many applications including medicine. Yet it evades consistent theoretical description. Here we study the effect of Hofmeister anions on gramicidin A channels in lipid membranes. Counterintuitively, we find that conductance of this perfectly cation-selective channel increases about two-fold in the H2PO4−<Cl−≈Br−≈NO3−<ClO4−<SCN− series. Channel dissociation kinetics show even stronger dependence, with the dwell time increasing ~20-fold. While the conductance can be quantitatively explained by the changes in membrane surface potential due to exclusion of kosmotropes from (or accumulation of chaotropes at) the surface, the kinetics proved to be more difficult to treat. We estimate the effects of changes in the energetics at the bilayer surfaces on the channel dwell time, concluding that the change would have to be greater than typically observed for the Hofmeister effect outside the context of the lipid bilayer., Ion specificity and, in particular, the distinctive effects of anions in salt-induced protein precipitation have been known since the 1880’s, when Franz Hofmeister established the ranking of anions in their ability to regulate egg yolk protein water solubility []. Experimental and theoretical studies have given a detailed empirical picture of the phenomenon, the nature of the ionic interactions with the surfaces leading to the Hofmeister effect is still under debate []. The only consensus is that it cannot be explained by standard theories of electrolytes. For example, bromide is unique in that its salts were recognized as a drug to treat epilepsy a couple of dozen years before Hofmeister’s studies [] and they are still in use to treat specific types of refractory seizures in children [], but the mechanism of their action remains elusive., , Hofmeister effect studied with a nanopore in a neutral lipid membrane. Rather unexpectedly, we find that conductance of a purely cation-selective peptide pore is regulated by anions in correlation with their position in the Hofmeister series. Moreover, the pore conformational dynamics are highly sensitive to the anion species. We relate both effects to preferential depletion of kosmotropic anions (accumulation of chaotropic anions) at the membrane-water interface

Correlations of Specific Ionic Effects using Ion Channels and Surface Charge Measurements

poster abstractSpecific ionic effects, as captured in the Hofmeister series, have been observed in many biological phenomena including protein folding and aggregation and lipid bilayer interactions. Previously we have shown that the Hofmeister effect is present in the activity of gramicidin A channels. In particular, measurements of channel open lifetime and conductance in potassium salts clearly show the existence of two distinct ionic classes that could be identified as kosmotropic and chaotropic. To further investigate this behavior, we have measured the zeta potential of diphytanoyl phosphatidylcholine (DPhPC) liposomes in salt solutions. We observe that anions alter the surface charge of the liposomes depending on the classification of the anion as kosmotropic or chaotropic. Chaotropic anions (SCN-, ClO4-) decrease the surface charge of the liposomes while kosmotropic anions (Cl-, H2PO4-, SO42-) have the opposite effect. These results correlate with our previous studies of cation conductance through gramicidin A channels adding new insight into ionic interactions at the lipid-water interface

Conductance of Ideally Cation Selective Channel Depends on Anion Type

poster abstractGramicidin A (gA) is a transmembrane, cation selective ion channel that has been used in many biophysical studies of lipid bilayers, in particular for investigations of lipid-protein interactions and membrane electrostatics. In addition, it was found that ionic interactions with neutral lipid membranes also affect the kinetics of gA channels. Here we report measurements of gA ion-channels for a series of sodium and potassium salts that show an anion-dependence of gA conductance. We find that gA conductance varies significantly with the anion type with ClO4 and SCN producing distinctly larger conductance values than Cl, F, and H2PO4. These results can provide new insights into ion-lipid membrane interactions and ion channel functions in general